The overall goal of the proposed research is to empower NMR spectroscopy for characterizing large and biologically significant proteins and protein complexes. This will lead to new insights into biological mechanisms and will ultimately lead to improving human health. Proteins function primarily by interacting with other proteins, nucleic acids, small ligands or substrates. Thus, the overall goal of this grant is to understand mechanism based on structural insights, with a focus on protein interactions. This will be achieved with developing a set of NMR methods that can characterize challenging protein complexes in the range of 50 kDa and beyond. All components of the proposal contain both technology developments and applications to important targets that play roles in disease, are potential drug targets, or producers of natural products. Component 1 (Wagner) is on NMR approaches for structural characterization of large proteins and protein complexes. It will develop new methods for studies of proteins and protein complexes and will apply these for characterizing interactions of a viral internal ribosome entry site (IRES) RNA with translation initiation factors. Component 2 (Wagner/Walsh) is on the Enterobactin non-ribosomal peptide synthetase. It will solve structures of large mufti-domain units of this system and determine structures of complexes between EntF and EntB. Component 3 (Reinherz) will elucidate the structure and function of the signaling elements of the T-cell receptor. Component 4 (Naar) will investigate the structure and function of the pleiotropic drug resistance system of pathogenic yeast. The research will be supported by three cores for Administration (Core A), NMR instrumentation and Chemistry (Core B) and Computation (Core C). The interaction between the research components and cores of this POI grant simulated the development of many new NMR and data processing techniques that now facilitate structural studies of challenging macromolecular systems. We anticipate that the proposed research will continue to advance the capabilities of NMR for structural biology.